Network pharmacological analysis on the mechanism of Linggui Zhugan decoction for nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD), represents a chronic progressive disease that imposes a significant burden on patients and the healthcare system. Linggui Zhugan decoction (LGZGD) plays a substantial role in treating NAFLD, but its exact molecular mechanism is unknown. Using network pharmacology, this study aimed to investigate the mechanism of action of LGZGD in treating NAFLD. Active ingredients and targets were identified through the integration of data from the TCMSP, GEO, GeneCards, and OMIM databases. Cytoscape 3.9.1 software, in conjunction with the STRING platform, was employed to construct network diagrams and screen core targets. The enrichment analysis of gene ontology and the Kyoto Encyclopedia of Genes and Genomes pathways were conducted by using the R. Molecular docking of the active ingredients and core targets was performed with AutoDock Vina software. We obtained 93 and 112 active ingredients and potential targets using the bioinformatic analysis of LGZGD in treating NAFLD. The primary ingredients of LGZGD included quercetin, kaempferol, and naringenin. The core targets were identified AKT1, MYC, HSP90AA1, HIF1A, ESR1, TP53, and STAT3. Gene ontology function enrichment analysis revealed associations with responses to nutrient and oxygen levels, nuclear receptor activity, and ligand-activated transcription factor activity. Kyoto Encyclopedia of Genes and Genomes signaling pathway analysis implicated the involvement of the PI3K-Akt, IL-17, TNF, Th17 cell differentiation, HIF-1, and TLR signaling pathways. Molecular docking studies indicated strong binding affinities between active ingredients and targets. LGZGD intervenes in NAFLD through a multi-ingredient, multi-target, and multi-pathway approach. Treatment with LGZGD can improve insulin resistance, oxidative stress, inflammation, and lipid metabolism associated with NAFLD.


Introduction
Nonalcoholic fatty liver disease (NAFLD) is a liver disorder characterized by hepatic steatosis exceeding 5%, without secondary causes. [1]NAFLD prevalence is rising worldwide, with a prevalence of 30% in China and 25% in the world. [2,3]AFLD is a major cause of hepatic sclerosis, liver cancer, and SG and LW contributed equally to this work.Dang [2022] No. 24), Huang Guihua TCM Master Training Project (Guizhong medical science and education [2022] No. 6), Innovation Project of Guangxi Graduate Education (YCBZ2023148), and Huang Guihua Guangxi Famous Traditional Chinese Medicine Inheritance Studio (Guiwei Traditional Chinese Medicine [2017] No. 2).

The authors have no conflicts of interest to disclose.
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.This study is a summary of data from published articles and does not address issues related to patient ethics, etc.Therefore, this study does not require approval by the ethics committee.
[6] A recent global burden of disease study reveals that NAFLD causes a heavy burden in Asia, the Middle East, and North Africa, accounting for about half of the global burden of hepatic sclerosis and hepatoma caused by NAFLD. [7]No specific pharmacological treatment is available for NAFLD.The primary methods include a low-calorie diet, exercise therapy, and treatment of its complications; however, patients' compliance is poor, and the curative effect is limited. [8]To effectively address NAFLD, the development of targeted pharmaceuticals is imperative to enhance treatment options.
In the treatment of chronic diseases, traditional Chinese medicine (TCM) presents significant advantages, particularly in the context of liver diseases.TCM employs tailored treatments  based on specific diagnoses for different diseases. [9,10]NAFLD is classified under the TCM category of "Xie-Tong," "Gan-Pi," "Fei-Qi," and "Ji-Ju."TCM has been widely recognized for its efficacy in preventing and treating NAFLD, offering precise therapeutic effects with minimal adverse reactions. [11]Hui D [12] reported that a spleen-strengthening and liver-draining formula demonstrated the capacity to reduce AST, ALT, and liver fat content in NAFLD patients by regulating intestinal flora.Shuganzhi Tablet effectively treated NAFLD, while hesperidin, polydatin, and naringin may be its most effective components. [13]inggui Zhugan decoction (LGZGD), derived from the Chinese medical text "Treatise on Cold and Miscellaneous Diseases," is composed of Fu Ling (Poria cocos), Gui Zhi (Cinnamomi ramulus), Bai Zhu (Atractylodes macrocephala), and Gan Cao (Glycyrrhiza uralensis).Fu Ling, the primary drug in this prescription, invigorates the middle energizer, removes dampness, and promotes diuresis.Gui Zhi, functioning as an assistant, contributes by warming the spleen yang and enhancing diuresis.Bai Zhu has the functions of invigorating the spleen and Qi, drying dampness,    and diuresis.Gan Cao has the curative effect of Tonifying Qi and harmonizing various drugs.Combining the 4 drugs has the function of Warming Yang and promoting diuresis.Currently, the pharmacological research of LGZGD primarily involves antioxidant, anti-inflammatory, regulation of lipid metabolism, insulin resistance, and liquid metabolism. [14]High-quality articles exploring the mechanism of the efficacy of LGZGD in treating NAFLD remain scarce, despite supportive findings from clinical observational studies. [15,16]Network pharmacology is a new technology that can systematically study the possible mechanism of drugs on diseases by constructing the interaction network of "drugsdiseases-targets-pathways" and provide new insights into how TCM treats diseases.We aim to study the effective components, targets, biological processes (BPs), and pathways of LGZGD in treating NAFLD using network pharmacological methods to provide new ideas for further study.

Screening of LGZGD's active ingredients and targets
The TCM systematic pharmacology database (TCMSP, https:// old.tcmsp-e.com/tcmsp.php) was used to retrieve each TCM in LGZGD.The LGZGD active ingredients were screened form oral bioavailability ≥ 30% and drug-like ≥ 0.18.Then, the "Related Targets" option was selected to find all prediction targets associated with each active ingredient.All the screened targets were imported into UniProt (https://www.uniprot.org)database because of a nonstandard problem with searching targets in TCMSP database.The obtained target name was transformed into the target protein's gene name.Simultaneously, the input target protein name was called the restricted species "Homo sapiens," so all protein names were corrected.

Screening of NAFLD disease-related targets
A search for the keyword "nonalcoholic fatty liver disease" was conducted in Gene Expression Omnibus (GEO, https:// www.ncbi.nlm.nih.gov/),GeneCards (https://www.genecards.org/), and Online Mendelian Inheritance in Man (OMIM, https://omim.org/).The results of search in the 3 databases were summarized and de-duplicated to identify NAFLD diseaserelated target.The NAFLD (GSE89632) expression profile was downloaded from the GEO.The GSE89632 dataset included 20 NAFLD samples and 24 healthy samples on the GPL14951 platform.The differentially expressed genes (DEGS) were obtained under |logFC| > 1 and P.adj < 0.05 and displayed as the heat map.

TCM-active ingredients-potential targets network construction
The common target genes shared between LGZGD and NAFLD were identified using the "Venn" R package, signifying potential treatment targets.Subsequently, to construct a comprehensive network depicting the relationship among "TCMactive ingredients-potential targets," we generated network and type attribute files.These files were then imported into Cytoscape 3.9.1 software.The degree value of active ingredients was calculated using the "Network Analyzer" tool within Cytoscape 3.9.1 software.

Protein-protein interaction (PPI) network graph construction
The common targets were entered into the STRING database (https://string-db.org/) to construct the PPI network under the conditions of species selection "Homo sapiens," "hide disconnected nodes in the network," and "highest confidence (>0.90)."For further analysis, the ".TAV" file format of the PPI network information is uploaded to Cytoscape 3.9.1 software.Cytoscape's CytoNCA tool was used for topology analysis to screen the core targets according to the ranking of the values of Degree, Betweenness centrality, Closeness centrality, Eigenvector centrality, Network centrality, and Local Average Connectivitybased method.

Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis
R (version 4.2.2) package "clusterProfiler" was used to analyze the common targets, including GO and KEGG enrichment analysis.

Molecular docking
This study investigated molecular docking for analyzing the characteristics of receptors and the formation of molecular complexes between receptors and small drug molecules via computer space and energy matching and to predict the operation process of complex structures.The ID and 3D model structure of the protein corresponding to core target, as a macromolecular receptor, was retrieved from the PDB database (http://www.rcsb.org/).Then, the water molecular structure and original small molecule ligands of the core target protein were removed using PyMOL 2.4.1 software.From PubChem database (https://pubchem.ncbi.nlm.nih.gov/), the 2D structure of the core active ingredient was downloaded as a small molecule ligand.The 2D structure of the active ingredient was added to Chem3D software, converted into a 3D structure that optimizes the minimized energy, and exported as a ".Mol2" format.Protein and active ingredients were pretreated using AutodockTools 1.5.6 and converted to ".pdbqt" format, and then they were docked using Autodockvina 1.1.2.Display of docking results was performed with PyMOL software.

LGZGD active ingredient and target information
We obtained 7 active ingredients from Bai Zhu, 15 from Fu Ling, 92 from Gan Cao, and 7 from Gui Zhi.Among them, sitosterol is the common active ingredient from Gan Cao and Gui Zhi, so LGZGD has 120 active ingredients.We retrieved 249 targets corresponding to 120 active ingredients of LGZGD from the TCMSP database.After combined data deduplication and target protein name correction using the UniProt database, 227 active targets were included, corresponding to 103 active ingredients.

NAFLD disease target data
We obtained 409 DEGS, including 135 up-regulated, from the GSE89632 dataset (Fig. 1).Additionally, 1227 and 365 disease targets were obtained in Genecards and OMIM databases, respectively.After the 3 database results were combined and de-duplicated, 1862 NAFLD disease targets were obtained (Fig. 2A).

TCM-active ingredients-potential targets network construction
LGZGD exhibits 112 potential targets associated with 93 active ingredients in the treatment of NAFLD, as determined using R language software (4.2.2) (Fig. 2B).Then, the targets and ingredients were uploaded to Cytoscape 3.9.1 software for building a comprehensive "TCM-active ingredients-potential targets" network (Fig. 3).The circle in the network diagram represents TCM active ingredients, different colors represent TCM sources (orange represents Gan Cao, red represents Fu Ling, green represents Bai Zhu, blue represents Gui Zhi), and sky blue rectangular nodes represent potential targets.Nodes with higher degree values may signify key ingredients or targets of LGZGD in the treatment of NAFLD.The Network Analyzer results indicated that the top 3 active ingredients of the degree value are quercetin, kaempferol, and naringenin, which may be LGZGD's key ingredients for treating NAFLD (Table 1).

Construction and analysis of the PPI network
The common targets were imported into the STRING database to create the PPI network diagram of potential targets of LGZGD for treating NAFLD (Fig. 4).The values of Degree, Betweenness centrality, Closeness centrality, Eigenvector centrality, Network centrality, and Local Average Connectivity-based method of the target were analyzed using the CytoNCA plug-in.After the first screening with these 6 values greater than the median, the second screening was conducted according to the same screening conditions.The core target was obtained via 2 screenings (Fig. 5).The core targets are serine/threonine kinase 1 (AKT1), myelocytomatosis (MYC), heat shock protein 90 alpha family class A member 1 (HSP90AA1), hypoxia-inducible factor-1alpha (HIF1A), estrogen receptor 1 (ESR1), cell tumor antigen p53 (TP53), and signal transducer and activator of transcription 3 (STAT3) (Table 2).

Enrichment analysis
The GO analysis presented that 2359 items were screened (P < .05).After ranking by P-value, the top 10 items for BP, cellular component, and molecular function were filtered to create a bar chart (Fig. 6 and Table 3).BP primarily involves the response to nutrient levels, xenobiotic stimulus, oxygen levels, lipopolysaccharide, decreased oxygen levels, oxidative stress, a molecule of bacterial origin, hypoxia, metal ion, and radiation.Cellular component was primarily enriched in membrane raft, membrane microdomain, RNA polymerase II transcription regulator complex, collagen-containing extracellular matrix, vesicle lumen, endoplasmic reticulum lumen, external side of the plasma membrane, plasma membrane raft, serine-type peptidase complex, and cytoplasmic vesicle lumen.Molecular function primarily included nuclear receptor activity, ligand-activated transcription factor activity, transcription coregulatory factor binding, DNA-binding transcription factor binding, cytokine activity, RNA polymerase II-specific DNA-binding transcription factor binding, protease binding, cytokine receptor binding, signaling receptor activator activity, and heme binding.KEGG data analysis obtained 158 related pathways (P < .05),including PI3K/Akt, IL-17, TNF, Th17 cell differentiation, HIF-1, and TLR signaling pathway (Fig. 7 and Table 4).

Molecular docking results
We docked 3 key active ingredients with targets (AKT1, MYC, HSP90AA1, HIF1A, ESR1, TP53, and STAT3).If the binding energy between ligand and receptor is ≤−5 kcal•mol −1 , it indicates that a small molecule ligand can bind to a protein receptor efficiently.The binding ability increased with lower binding energy.The results displayed that the binding energies of key active ingredients and core target docking were ≤−5 kcal•mol −1 (Table 5).To provide visual insights into the binding activities, we employed PyMOL software to visualize the molecular docking results of naringenin-ESR1 and quercetin-STAT3, as these interactions exhibited the strongest binding activity (Fig. 8).

Discussion
Current epidemiological studies have discovered that the rapid development of economic levels has led to changes in people's eating habits and pace of life, and the prevalence of NAFLD has increased yearly. [17]The NAFLD pathogenesis is unclear, primarily related to insulin resistance, abnormal lipid metabolism, oxidative stress, inflammatory response, and immune regulation imbalance. [18]TCM prescription is composed of different Chinese medicines with the distinct benefits of multi-ingredient, multi-target, and multi-pathway and helps treat NAFLD with complex pathogenesis.The application of network pharmacology represents a cutting-edge approach in elucidating the mechanisms of TCM prescription interventions in diseases.Therefore, this study used it to explore the mechanism of LGZGD in improving NAFLD, to find a new target for drug treatment of NAFLD, and provide a reference for improving the clinical efficacy and drug safety.According to network pharmacological analysis, LGZGD may treat NAFLD via quercetin, kaempferol, naringenin, and other major active ingredients.Molecular docking results also exhibited that them had good binding ability with multiple key targets.Recent studies have increasingly highlighted the potential of dietary polyphenolic compounds as therapeutic agents or nutritional supplements for various liver diseases. [19]he main groups of polyphenols include flavonoids, phenolic acids, lignands, stilbenes, and curcuminoids. [20][23] The flavonoids identified in this study, namely, quercetin, kaempferol, and naringenin, offer promising advantages in NAFLD treatment through various mechanisms, which is consistent with findings from other studies. [24]Many in vivo and in vitro experimental evidence have identified various molecular mechanisms of quercetin in preventing the occurrence and progress of NAFLD, including antioxidant stress, anti-inflammatory, improving fatty acid metabolism, and regulating gut microbiota and bile acid metabolism. [25]Quercetin has therapeutic potential for NAFLD-related learning and memory disorders by regulating glucose and lipid metabolic dysfunction, balancing the protein expression of synaptic plasticity markers, and triggering receptors expressed on myeloid cells-1/2 in the hippocampus. [26]Quercetin can prevent NAFLD by regulating mitochondrial ROS-mediated ferroptosis and improving liver lipid toxicity and accumulation. [27]Through multi-omics research, Lu [28] found that kaempferol can improve lipid metabolism, oxidative reaction and inflammation in treating nonalcoholic steatohepatitis (NASH) mice.Kaempferol and kaempferoside can reduce lipid accumulation and oxidative stress in HepG2 cells induced by oleic acid, potentially benefiting NAFLD treatment. [29]Naringenin inhibits NLR family proteins containing Pyrin-related domain 3/nuclear factor-kappaB Signaling pathway alleviates inflammation in the mice liver to prevent NAFLD. [30]Naringenin alleviates NAFLD by directly and indirectly activating adenosine monophosphate-activated protein kinase to increase energy expenditure and regulate autophagy. [31]Naeini F [32] discovered that naringenin could regulate energy balance, glucose and lipid metabolism, inflammation, and oxidative stress and reduce triglyceride accumulation in hepatocytes.Therefore, LGZGD may play the role of antioxidant stress, antiinflammatory, improving lipid metabolism, and regulating intestinal microbiota in treating NAFLD via quercetin, kaempferol, naringenin, and other active ingredients.
According to the network diagram of "TCM-active ingredientspotential targets" and PPI network topology analysis, the key potential targets are AKT1, MYC, HSP90AA1, HIF1A, ESR1, TP53, and STAT3.Genetic variations in AKT1 have been associated with NAFLD risk. [33]Forkhead box protein O1 (FoxO1) is the main target of insulin action, possibly leading to lipid and glucose metabolism disorders after being activated. [34]diponectin can improve NAFLD by inhibiting the FoxO1 expression via AKT1/FoxO1 signaling pathway. [35]In a study by Jiang, [36] that an NAFLD mouse model expressed lower www.md-journal.comlevels of AKT1 and higher mitogen-activated protein kinase 1, caspase 3, p53, and vascular Endothelial Growth Factor A compared to those observed in the normal group.MYC is crucial for cell proliferation.C-MYC signal transduction is associated with NAFLD-related fibrosis. [37]he specific reduction of MYC in the mouse intestine can improve HFD-induced obesity, insulin resistance, liver steatosis, and steatohepatitis. [38]Peroxisome proliferator-activated receptor-γ (PPARγ) is involved in the NAFLD pathogenesis via adipogenesis, insulin resistance, inflammation, and oxidative stress. [39]HSP90AA1 is a common subtype of HSP90.HSP90 can regulate the activity of PPARγ in NAFLD, which is of great significance for treating NAFLD. [40]HIF-1α is a key regulatory factor that plays an active role in hypoxia.HIF-1α can increases the expression of interleukin-1 beta (IL-1β) by mediating autophagy injury of macrophages, thus promoting the occurrence of NASH. [41]Hypoxia can induce the upregulation of HIF-1α, activating hepatic stellate cells, which ultimately results in hepatic fibrosis. [42]Yexiaodan [43] discovered that inhibiting the HIF-1α/PPARγ signaling pathway can decrease inflammation and steatosis.The ESR1 expression is lower in men than women, but ESR1 knockout will cause insulin resistance phenotype, presenting obesity, glucose intolerance, liver steatosis, and metabolic inflammation of adipose tissue and liver. [44]P53 regulates various metabolic pathways, including lipid metabolism, and regulates lipid synthesis, transport, and decomposition via multiple pathways. [45]Upregulation of the p53 pathway can accelerate NAFLD development by promoting hepatocyte senescence and apoptosis. [46]Silencing functional p53 can alleviate NAFLD by promoting autophagy induced by high mobility group box-1 protein. [47]In addition to its involvement in cell proliferation, STAT3 is closely associated with liver damage, inflammation, regeneration, and cancer. [48]A significant correlation exists between STAT3 activation and the severity of hepatic fibrosis in patients with NAFLD. [49]It is speculated that LGZGD can intervene in insulin resistance, inflammation, oxidative stress, and lipid metabolism of NAFLD via the above targets to achieve the therapeutic purpose.LGZGD is primarily associated with PI3K/Akt, IL-17, TNF, Th17 cell differentiation, HIF-1, and signaling pathways in treating NAFLD.PI3K/Akt signaling pathway is the key pathway of insulin information transmission, and its signal transmission disorder easily leads to insulin resistance, assisting in the development of NAFLD. [50]Activation of PI3K/Akt-nuclear factor E2 related factor 2-antioxidant response element pathway can improve liver oxidative stress and inflammatory response. [51]By stimulating autophagy, the inhibition of the PI3K/Akt/mammalian target of the rapamycin signaling pathway can alleviate lipid accumulation, inflammation and NAFLD. [52]Th17 cells are an important subset of CD4 + lymphocytes that participate in various inflammatory and immune diseases and can induce epithelial cells to secrete interleukin-6, IL-17, TNFα, and other proinflammatory factors, thus promoting hepatocyte inflammation and steatosis. [53]As the primary effector of Th17 cells, IL-17 deficiency can interfere with intestinal microbiota composition in methionine and choline-deficient diet-fed mice, such as increased pathogenic bacteria abundance and decreased probiotics, aggravate the liver steatosis accumulation and promote the NAFLD/NASH progress. [54]Among the many pro-inflammatory factors that contribute to the pathogenesis of NASH, TNFα has a crucial role in many aspects of NASH progress. [55]HIF-1 mediates liver fibrosis development in the NAFLD mouse model. [56]TLR is widely expressed in hepatocytes, Kupffer cells, and hepatic satellite cells. [57]The majority of TLRs induce pro-inflammatory and pro-fibrotic cytokines, participating in the progression of NAFLD. [58]almitic acid-induced hepatocyte lipotoxicity is mediated by the activation of TLR4 inositol-dependent kinase 1α pathway. [59]Conversely, inhibition of TLR2 improves lipopolysaccharide-induced lipid accumulation and toxicity in hepatocytes. [60]ocking experiments confirmed that quercetin, kaempferol, and naringenin exhibit strong binding affinity with core targets (AKT1, MYC, HSP90AA1, HIF1A, ESR1, TP53, and STAT3), with binding energies less than −5 kcal•mol -1 .This suggests that the therapeutic potential of LGZGD in treating NAFLD may be attributed to the efficacy of these 3 key active ingredients.

Conclusion
This study concludes that LGZGD may have multi-ingredient, multi-target, and multi-pathway effects in treating NAFLD, possibly playing a therapeutic role by improving insulin resistance, antioxidant stress, anti-inflammatory, lipid metabolism, and other mechanisms.The results exhibited that quercetin, kaempferol, and naringenin were the primary active compounds in LGZGD.Meanwhile, AKT1, MYC, HSP90AA1, HIF1A, ESR1, TP53, and STAT3 are the potential therapeutic targets of LGZGD in treating NAFLD.LGZGD can treat NAFLD by regulating PI3K/Akt, IL-17, TNF, and other signaling pathways.This study provides a new idea, direction, and reference for the research of LGZGD for treating NAFLD and a theoretical basis for developing new drugs for LGZGD.

Figure 3 .
Figure 3. TCM-active ingredients-potential targets network.The circle in the network diagram represents TCM active ingredients, different colors represent TCM sources (orange represents Gan Cao, red represents Fu Ling, green represents Bai Zhu, blue represents Gui Zhi), and sky blue rectangular nodes represent potential targets.TCM = traditional Chinese medicine.

Figure 5 .
Figure 5. Screening process for core targets of LGZGD in treating NAFLD.(A) Filtered for the first time core network diagram, (B) filtered for the second time core network diagram, (C) streamlined core network, LGZGD = Linggui Zhugan decoction, NAFLD = nonalcoholic fatty liver disease.

Figure 6 .
Figure 6.GO function enrichment analysis of targets of LGZGD in treating NAFLD.BP = biological process, CC = cellular component, GO = gene ontology, MF = molecular function.

Table
Core active ingredients of LGZGD in treating NAFLD.

Table 2
Seven core targets of LGZGD in treating NAFLD.

Table 3
The top 10 GO enrichment items of targets of LGZGD in treating NAFLD.

Table 4
The top 30 KEGG enrichment items of targets of LGZGD in treating NAFLD.

Table 5
Binding energy of active ingredients to core targets.